Modified Latency Associated Protein Construct

ABSTRACT

The present invention provides a fusion protein comprising a latency associated peptide (LAP), a pharmaceutically active agent and an amino acid sequence comprising a dimerisation domain, wherein the LAP and the pharmaceutically active agent are connected by an amino acid sequence comprising a proleolytic cleavage site. Also provided are nucleic acids enclosing such fusion proteins, process for their preparation, pharmaceutical compositions, kits and uses thereof in medicine.

The present invention relates to the use of proteins, proteinderivatives and DNA constructs that confer latency to pharmaceuticallyactive agents. The present invention also relates to improved methods ofproviding latency to pharmaceutically active agents.

Most cytokines and growth factors are expressed under tight controlmechanisms. Their gene expression is regulated by environmental stimulisuch as infection, cell-cell interactions, change in extracellularmatrix composition and interactions with adhesion molecules or viastimulation with other cytokines.

In addition to the control at the transcriptional andpost-transcriptional level, some cytokines are not released into themedium unless a second signal activates the cell. A third level ofregulation for cytokine activity is found in molecules which aresecreted in a latent form and become “activated” by releasing thecytokine moiety where processes of inflammation, wound healing andtissue repair takes place (Khalil N, Microbes and Infection, 1,1255-1263 (1999). In this latter respect, transforming growth factorbeta (TGFβ) has received greatest attention.

TGFβ is synthesized as a dimeric latent cytokine composed of an aminoterminal latency associated protein (LAP) and the active TGFβ cytokineat its COOH terminal end (Roberts and Sporn, Peptide Growth Factors andtheir Receptors: Sporn, M B and Roberts, AB, Springer-Verlag, 419-472(1996); Roth-Eicchorn et al., Hepatology, 28 1588-1596 (1998)). Theprecursor peptide contains a signal peptide (residues 1-29) necessaryfor protein secretion and guiding the molecule through the Golgiapparatus to become processed by proteolytic cleavage and glycosylation.The LAP domain is separated from TGFβ by proteolytic cleavage atarginines (277-278). Mature TGFβ begins at alanine 279. The LAP, inaddition to protect TGFβ, contains important residues necessary for theinteraction with other molecules. Mutations in the LAP domain haverecently been associated with the autosomal dominant Camurati-Engelmanndisease (Janssens et al., Nature Genetics, 26, 273-275 (2000). Cysteines224 and 226 are important in the intermolecular disulphide bond betweentwo LAPs. Their mutation to serine renders the molecule “active”(Sanderson et al., Proc. Natl. Acad. Sci. USA, 92, 2572-2576 (1995);Brunner et al., Mol. Endocrinol. 6, 1691-1700 (1992); Brunner et al., J.Biol. Chem, 264, 13660-13664 (1989)). The RGD motif (245-247)facilitates the interaction with integrins (Munger et al., Mol, Biol. ofthe Cell, 9, 2627-2638 (1998; Derynck R, TIBS, 19, 548-553 (1994)).Nucleic acid encoding TGFβ is described in U.S. Pat. No. 5,801,231.

In most cell types studied, including those of mesenchymal, epithelialand endothelial origin, TGFβ is secreted in a latent form consisting ofTGFβ and its latency associated peptide (LAP) propeptide dimers,covalently linked to latent TGFβ-binding proteins (LTBPs). LTBPs arealso needed for the secretion and folding of TGFβ (Miyazano et al., EMBOJ. 10, 1091-1101 (1991); Miyazano et al., J. Biol. Chem. 267, 5668-5675(1992); Eklov et al., Cancer Res. 53, 3193-3197 (1993)). Cysteine 33 isimportant for the disulphide bridge with the third 8 cysteine-richrepeat of latent TGFβ binding protein (LTBP) (Saharinen et al., The EMBOJournal, 15, 245-253 (1996). Modification of LTBP by enzymes such asthrombospondin (Schultz et al., The Journal of Biological Chemistry,269, 26783-26788 (1994); Crawford et al., Cell, 93, 1159-1170 (1998)),transglutaminase (Nunes et al., J. Cell, Biol. 136, 1151-1163 (1997);Kojima et al., The Journal of Cell Biology, 121, 439-448 (1993)) andMMP9, MMP2 (Yu and Stamenkovic, Genes and Dev, 14, 163-176 (2000)) couldrelease the active portion of TGFβ from the latent complex.

Cytokines are natural products serving as soluble local mediators ofcell-cell interactions. They have a variety of pleiotropic actions, someof which can be harnessed for therapeutic purposes. Targeting ofcytokines to specific cell types using scFv (Lode et al., Pharmacol.Ther, 80, 277-292 (1998)) and vWF (Gordon et al., Human Gene Therapy, 8,1385-1394 (1997)) have focused entirely on the active cytokine moiety ofthe cytokine complex.

Pharmacologically active proteins or other medicines based on suchagents, which have to be administered at very high concentrationssystemically in order to achieve biologically effective concentrationsin the tissue being targeted, tend to give rise to undesirable systemiceffects, for example toxicity, which limit their use and efficacy.

The principles underlying the construction of such a system forproviding latency to pharmaceutically active agents using the LAP ofTGF-β was described in WO 02/055098 and WO 2009/077755. In the naturallyoccurring LAP-TGF-β complex, the latency associated peptide forms aprotective shell around TGF preventing it from being degraded. Theclosed nature of the shell is guaranteed because of internalinteractions between TGF with LAP. These interactions are notnecessarily expected when the ‘payload’ is another cytokine, growthfactor or peptide or pharmaceutically active compound, and may result ina permeable shell which allows entry of mobile target molecules into theshell. For example, if the target molecule is a soluble receptor, thereceptor may be able to enter the shell and interact with thepharmaceutically active agent even when the pharmaceutically activeagent is bound to the LAP. This could lead to off-site activity whichmay have undesirable side effects. Furthermore, while improving proteinproduction utilizing suspension cell cultures according to WO2009/077755, the inventors found that, along with dimers, the LAP fusionproteins also tended to form active monomers.

The present inventors have now developed an improved means for providingpharmaceutically active agents in latent form based on this system.

According to the first aspect of the invention there is provided afusion protein comprising a latency associated peptide (LAP), apharmaceutically active agent and an amino acid sequence comprising adimerisation domain wherein the LAP and pharmaceutically active agentare connected by an amino acid sequence comprising a proteolyticcleavage site.

The fusion protein comprising a LAP, a proteolytic cleavage site, apharmaceutically active agent and a dimerisation domain may provide forsite specific activation of the latent pharmaceutically active agent.The term “site specific activation” as used herein means, in generalterms and not limited to the removal or reduction of latency, conferredon a pharmaceutically active agent, by site-specific cleavage at theproteolytic cleavage site.

Site-specific cleavage at the proteolytic cleavage site is expected totake place concomitantly with the restored activation of thepharmaceutically active agent.

The term “latent pharmaceutically active agent” as used herein mayinclude, but is not limited to, pharmaceutically active agents which arelatent due to their association with LAP and a proteolytic cleavagesite. Specifically, the pharmaceutically active agent may be latent byvirtue of its fusion to a LAP associated proteolytic cleavage site toform a latent fusion protein. The pharmaceutically active agent may beof natural or synthetic origin.

The fusion protein may be constructed as shown in FIG. 4(c) in which thedimerisation domain is fused to the LAP. An additional proteolyticcleavage site and/or linker sequence may be inserted also between thedimerisation domain and the LAP. A secretory signal peptide (i.e. theprecursor peptide) may be fused to the dimerisation domain also at theN-terminal of the dimerisation domain.

The term “protein” in this text means, in general terms, a plurality ofamino acid residues joined together by peptide bonds. It is usedinterchangeably and means the same as peptide, oligopeptide, oligomer orpolypeptide, and includes glycoproteins and derivatives thereof. Theterm “protein” is also intended to include fragments, analogues andderivatives of a protein wherein the fragment, analogue or derivativeretains essentially the same biological activity or function as areference protein.

The fragment, analogue or derivative of the protein as defined in thistext, may be at least 6, preferably 10 or 20, or up to 50 or 100 aminoacids long.

The fragment, derivative or analogue of the protein may be (i) one inwhich one or more of the amino acid residues are substituted with aconserved or non-conserved amino acid residue (preferably, a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorysequence which is employed for purification of the polypeptide. Suchfragments, derivatives and analogues are deemed to be within the scopeof those skilled in the art from the teachings herein.

Particularly preferred are variants, analogues, derivatives andfragments having the amino acid sequence of the protein in which severale.g. 5 to 10, or 1 to 5, or 1 to 3, 2, 1 or no amino acid residues aresubstituted, deleted or added in any combination. Especially preferredamong these are silent substitutions, additions and deletions, which donot alter the properties and activities of the protein of the presentinvention. Also especially preferred in this regard are conservativesubstitutions.

An example of a variant of the present invention is a fusion protein asdefined above, apart from the substitution of one or more amino acidswith one or more other amino acids. The skilled person is aware thatvarious amino acids have similar properties. One or more such aminoacids of a substance can often be substituted by one or more other suchamino acids without eliminating a desired activity of that substance.

Thus the amino acids glycine, alanine, valine, leucine and isoleucinecan often be substituted for one another (amino acids having aliphaticside chains). Of these possible substitutions it is preferred thatglycine and alanine are used to substitute for one another (since theyhave relatively short side chains) and that valine, leucine andisoleucine are used to substitute for one another (since they havelarger aliphatic side chains which are hydrophobic). Other amino acidswhich can often be substituted for one another include: phenylalanine,tyrosine and tryptophan (amino acids having aromatic side chains);lysine, arginine and histidine (amino acids having basic side chains);aspartate and glutamate (amino acids having acidic side chains);asparagine and glutamine (amino acids having amide side chains); andcysteine and methionine (amino acids having sulphur containing sidechains).

Substitutions of this nature are often referred to as “conservative” or“semi-conservative” amino acid substitutions.

Amino acid deletions or insertions may also be made relative to theamino acid sequence for the fusion protein referred to above. Thus, forexample, amino acids which do not have a substantial effect on theactivity of the polypeptide, or at least which do not eliminate suchactivity, may be deleted. Such deletions can be advantageous since theoverall length and the molecular weight of a polypeptide can be reducedwhilst still retaining activity. This can enable the amount ofpolypeptide required for a particular purpose to be reduced—for example,dosage levels can be reduced.

Amino acid insertions relative to the sequence of the fusion proteinabove can also be made. This may be done to alter the properties of asubstance of the present invention (e.g. to assist in identification,purification or expression, as explained above in relation to fusionproteins).

Amino acid changes relative to the sequence for the fusion protein ofthe invention can be made using any suitable technique e.g. by usingsite-directed mutagenesis. It should be appreciated that amino acidsubstitutions or insertions within the scope of the present inventioncan be made using naturally occurring or non-naturally occurring aminoacids. Whether or not natural or synthetic amino acids are used, it ispreferred that only L-amino acids are present.

A protein according to the invention may have additional N-terminaland/or C-terminal amino acid sequences. Such sequences can be providedfor various reasons, for example, glycosylation.

The term “fusion protein” in this text means, in general terms, one ormore proteins joined together by chemical means, including hydrogenbonds or salt bridges, or by peptide bonds through protein synthesis orboth.

The latency associated peptide (LAP) of the present invention mayinclude, but is not limited to, the coding sequence for the precursordomain of TGFβ or a sequence which is substantially identical thereto.

“Identity” as known in the art is the relationship between two or morepolypeptide sequences or two or more polynucleotide sequences, asdetermined by comparing the sequences. In the art, identity also meansthe degree of sequence relatedness (homology) between polypeptide orpolynucleotide sequences, as the case may be, as determined by the matchbetween strings of such sequences. While there exist a number of methodsto measure identity between two polypeptide or two polynucleotidesequences, methods commonly employed to determine identity are codifiedin computer programs. Preferred computer programs to determine identitybetween two sequences include, but are not limited to, GCG programpackage (Devereux, et al., Nucleic acids Research, 12, 387 (1984),BLASTP, BLASTN, and FASTA (Atschul et al., J. Molec. Biol. 215, 403(1990).

The LAP of the present invention may comprise the precursor domain ofTGFβ, for example, the precursor peptide of TGFβ-1, 2 or 3 (from human)(Derynck et al., Nature, 316, 701-705 (1985); De Martin et al., EMBO J.6 3673-3677 (1987); Hanks et al., Proc. Natl. Acad. Sci. 85, 79-82(1988); Derynck et al., EMBO J. 7, 3737-3743 (1988); Ten Dyke et al.,Proc. Natl. Acad. Sci. USA, 85, 4715-4719 (1988)) TGFβ-4 (from chicken)(Jakowlew et al., Mol. Endocrinol. 2, 1186-1195 (1988)) or TGFβ-5 (fromxenopus) (Kondaiah et al., J. Biol. Chem. 265, 1089-1093 (1990)). Theterm “precursor domain” is defined as a sequence encoding a secretorysignal peptide (i.e. a precursor peptide) which does not include thesequence encoding the mature protein. The amino acid sequences of theprecursor domain of TGFβ1, 2, 3, 4 and 5 (Roberts and Sporn, PeptideGrowth Factors and their Receptors: Sporn, M B and Roberts, AB,Springer-Verlag, Chapter 8, 422 (1996)) are shown in FIG. 1.

Preferably, the amino acid sequence of the LAP has at least 50%identity, using the default parameters of the BLAST computer program(Atschul et al., J. Mol. Biol. 215, 403-410 (1990) provided by HGMP(Human Genome Mapping Project), at the amino acid level, to theprecursor domain of TGFβ1, 2, 3, 4 or 5 (Roberts and Sporn, PeptideGrowth Factors and their Receptors: Sporn, M B and Roberts, AB,Springer-Verlag, Chapter 8, 422 (1996)) as shown in FIG. 1. Morepreferably, the LAP may have at least 60%, 70%, 80%, 90% and still morepreferably 95% (still more preferably at least 99%) identity, at thenucleic acid or amino acid level, to the precursor domain of TGFβ1, 2,3, 4 or 5 as shown in FIG. 1 which comprises residues 1 to 278.

The LAP may comprise the LAP of TGFβ1, 2, 3, 4, or 5 (Roberts and Sporn,Peptide Growth Factors and their Receptors: Sporn, M B and Roberts, AB,Springer-Verlag, Chapter 8, 422 (1996)) as shown in FIG. 1.

The LAP may contain at least two, for example at least 4, 6, 8, 10 or 20cysteine residues for the formation of disulphide bonds.

The LAP may also comprise a sequence which has at least 50%, 60%, 70%,80%, 90%, 95% or 99% identity with a LAP sequence of FIG. 1, using thedefault parameters of the BLAST computer program provided by HGMP,thereto.

The “dimerisation domain” refers to a peptide having affinity for asecond peptide, such that the two peptides associate under physiologicalconditions to form a dimer. The second peptide may be the same or adifferent peptide. The dimerisation domain may also refer topolypeptides. The peptides or polypeptides may interact with each otherthrough covalent and/or non-covalent association(s).

The dimerisation domain may be linked to the latency associated peptideby a linker. The linker size can be varied to vary the size of the shellin order to accommodate the pharmaceutically active agent. The linkerpeptide may comprise the amino acid sequence GGGGS (SEQ ID NO:135) or amultimer thereof (for example a dimer, a trimer, or a tetramer), asuitable linker may be (GGGGS)₃ (SEQ ID NO:136), or a sequence ofnucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99%identity, using the default parameters of the BLAST computer programprovided by HGMP, thereto.

Examples of dimerisation domains include antibody fragment polypeptidessuch as an immunoglobulin Fc polypeptide, an immunoglobulin hingepolypeptide, a CH3 domain polypeptide, a CH4 domain polypeptide, a CH1domain or CL domain polypeptide; a leucine zipper domain (e.g., ajun/fos leucine zipper domain, see, e.g., Kostelney et al., J. Immunol.,148:1547-1553, 1992; or a yeast GCN4 leucine zipper domain); anisoleucine zipper domain; a dimerising region of a dimerisingcell-surface receptor (e.g., interleukin-8 receptor (IL-8R); or anintegrin heterodimer such as LFA-1 or GPIllb/111a); a dimerising regionof a secreted, dimerising ligand (e.g., nerve growth factor (NGF),neurotrophin-3 (NT-3), interleukin-8 (IL-8), vascular endothelial growthfactor (VEGF), or brain-derived neurotrophic factor (BDNF); see, e.g.,Arakawa et al., J. Biol. Chem. 269:27833-27839, 1994, and Radziejewskiet al., Biochem. 32:1350, 1993); or a polypeptide comprising at leastone cysteine residue (e.g., one, two, or three to about ten cysteineresidues) such that disulfide bond(s) can form between the polypeptideand a second polypeptide comprising at least one cysteine residue.Suitably the dimerisation domain may be an Fc polypeptide.

An immunogobulin hinge polypeptide typically comprises a region which isrich in proline and cysteine amino acid residues. A common sequencemotif present in the hinge polypeptide region may be may be CPXCP (SEQID NO:137) where X can be another residue that does not interfere withdimerisation, for example proline (P), arginine (R) or serine (S). Thehinge region polypeptide may be from around 10 to 75 amino acidresidues. The hinge region polypeptide may contain a plurality ofcysteine-cysteine disulphide bonds, for example of from 2 to 15. Thehinge region polypeptide may comprise the sequence CPXCP where X can beanother residue that does not interfere with dimerisation, for exampleproline (P), arginine (R) or serine (S). A number of repeats of thesequence CPXCP may be present also, for example 2, 3, 4, or 5 repeats,or greater.

Wypych et al., J. Biol. Chem. 28316194-16205, 2008 defined the followinghinge region peptide sequences for IgG antibodies as shown in Table 1below:

TABLE 1 IgG subtype Core hinge sequences IgG1EPKSCDKTHTCPPCP (SEQ ID NO: 138) IgG2 ERKCCVECPPCP (SEQ ID NO: 139) IgG3ELKTPLGDTTHTCPRCP (SEQ ID NO: 140) (EPKSCDTPPPCPRCP)₃ (SEQ ID NO: 141)IgG4 ESKYGPPCPSCP (SEQ ID NO: 142)

The terms “antibody” and “immunoglobulin” are used hereininterchangeably. An antibody molecule is made up of two identical heavy(H) and two identical light (L) chains held together by disulphidebonds. Each heavy chain comprises an Fc polypeptide. The two Fcpolypeptides from the two heavy chains dimerise to form the Fc region ofthe antibody molecule. The term “Fc region” refers to the constantregion of an antibody excluding the first constant region immunoglobulindomain of the heavy chain (CH1) that interacts with the constant portionof the light chain (CL) forming a CH1-CL domain pair. Thus, Fc regioncomprises the last two constant region immunoglobulin domains (CH2 andCH3) of IgA, IgD, and IgG, and the last three constant regionimmunoglobulin domains of IgE and IgM (CH2, CH3 and CH4), Anypolypeptide of the various immunoglobulin constant domains may thereforebe used in accordance with the present invention as a dimerisationdomain.

Several antibody effector functions are mediated through the binding ofthe Fc region to Fc receptors (FcR) found on the surface of many cellsfor example lymphocytes, macrophages, natural killer cells, etc. FcRsare defined by their specificity for antibody isotypes. For example, Fcreceptors for IgG antibodies are referred to as FcγR.

IgG is also bound by the neonatal Fc receptor (FcRn). In humans, IgGexhibits a long serum half-life. Studies indicate that this is due tothe protective effect of FcRn which binds to the Fc region of IgG andprevents degradation by allowing intracellular recycling.

The Fc polypeptide may be selected to alter, e.g. increase or decreasethe half-life of the fusion protein. As used herein, the term“half-life” refers to a biological half-life of a particular polypeptideor protein in vivo. Half-life may be represented by the time requiredfor half the quantity administered to a subject to be cleared from thecirculation and/or other tissues in the animal. In an embodiment of theinvention the Fc polypeptide is an IgG Fc polypeptide.

IgG antibodies can be further subdivided into IgG1, IgG2, IgG3 and IgG4.In an embodiment of the invention the Fc polypeptide may be IgG1, IgG2,IgG3 and IgG4 polypeptide, for example an IgG1 polypeptide.

The Fc polypeptide may be selected to target the fusion protein tospecific tissues, for example the mucosa. The IgA antibody plays animportant role in mucosal immunity for e.g. in the respiratory tract andthe gastrointestinal mucosal lining. In its secretory form, IgA is foundin mucous secretions such as tears, saliva, colostrum, gastrointestinaland genitourinary fluids. IgA deficiency is associated with a number ofautoimmune diseases such as rheumatoid arthritis, systemic lupuserythematosus and immune thrombocytopenic purpura. IgA deficiency isalso associated with allergic diseases such as asthma. In anotherembodiment of the invention the Fc polypeptide may be an IgApolypeptide.

The Fc polypeptides may be derived from the same antibody isotype toform a homodimeric Fc region or from different antibody isotypes to forma heterodimeric Fc region. The Fc polypeptide may be a naturallyoccurring polypeptide or may be an engineered polypeptide.

The LAP may provide a protective “shell” around the pharmaceuticallyactive agent thereby shielding it and hindering, or preventing, itsinteraction with other molecules in the cell surface or moleculesimportant for its activity.

The dimerisation domain enables the fusion of the amino terminals of twolatency associated proteins thereby effectively closing the LAP shell.The dimerisation domains are therefore complementary and permitdimerisation to occur. The closure of the shell does not depend on theinteraction of LAP with the pharmaceutically active agent. The closurealso prevents monomer formation of the LAP fusion proteins.

Closure of the shell also prevents any interaction between thepharmaceutically active agent and its target molecule unless thepharmaceutically active agent is released from the LAP fusion protein byproteolytic activity. This ensures site-specific delivery of thepharmaceutically active agent and may reduce off-site activity.

In one alternative embodiment of the invention, therefore, there isprovided a protein construct comprising two fusion proteins as definedin the first aspect of the invention which are present as a dimer. Thedimer is composed of monomers of the first aspect of the invention whichmay be the same or different with respect to the latency associatedpeptide (LAP), the pharmaceutically active agent, and the amino acidsequence comprising a proteolytic cleavage site.

The dimer may therefore be composed of a first monomer and a secondmonomer, in which the first monomer comprises a latency associatedpeptide (LAP), a pharmaceutically active agent and an amino acidsequence comprising a dimerisation domain wherein the LAP andpharmaceutically active agent are connected by an amino acid sequencecomprising a proteolytic cleavage site, and a second monomer comprisinga latency associated peptide (LAP), a pharmaceutically active agent andan amino acid sequence comprising a dimerisation domain wherein the LAPand pharmaceutically active agent are connected by an amino acidsequence comprising a proteolytic cleavage site.

The present invention therefore provides a composition comprising twofusion proteins according to the first aspect, wherein the fusionproteins are associated at the dimerisation domain in each fusionprotein.

The proteolytic cleavage site may comprise any protease specificcleavage site. The proteolytic cleavage site may include, but is notlimited to, a matrix metalloproteinase (MMP) cleavage site, a serineprotease cleavage site, an aggrecanase cleavage site, a site cleavableby a parasitic protease derived from a pathogenic organism (Zhang etal., J. Mol. Biol. 289, 1239-1251 (1999); Voth et al., Molecular andBiochemical Parasitology, 93, 31-41 (1998); Yoshioka et al., FoliaPharmacologica Japonica, 110, 347-355 (1997); Tort et al, Advances inParasitology, 43, 161-266 (1999); McKerrow, International Journal forParasitology, 29, 833-837 (1999); Young et al., International Journalfor Parasitology, 29, 861-867 (1999); Coombs and Mottram, Parasitology,114, 61-80 (1997)) or a site cleavable by the proteins of the complementcascade (Carroll, Annu. Rev. Immunol. 16, 545-568 (1998); Williams etal., Ann. Allergy, 60, 293-300 (1988)).

Suitably, the proteolytic cleavage site of the fusion proteins of thepresent invention is cleaved when the fusion protein is located at orintroduced to a site of disease diagnosed as an inflammatory condition,e.g. arthritis, or cancer which can be characterized by inflammationand/or tissue remodelling.

A MMP cleavage site may comprise a number of amino acid residuesrecognisable by MMP. Preferably, the MMP cleavage site comprises theminimum number of amino acid residues required for recognition andcleavage by MMP. Moreover, the amino acids of the MMP site may be linkedby one or more peptide bonds which are cleavable, proteolytically, byMMP. MMPs which may cleave the MMP site include, but are not limited to,MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10 and MMP13 (Yu and Stamenkovic,Genes and Dev. 14, 163-176 (2000); Nagase and Fields, Biopolymers, 40,399-416 (1996); Massova et al., J. Mol. Model. 3, 17-30 (1997); reviewedin Vu and Werb; Genes and Dev. 14, 2123-2133 (2000)).

The MMP cleavage site e.g. any one or more of MMP1, MMP2, MMP3, MMP7,MMP8, MMP9 or MMP10 may be as shown in FIG. 2 or a sequence of aminoacids which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99% identitywith the sequences shown in FIG. 2, using the default parameters of theBLAST computer program provided by HGMP, thereto. In an embodiment theMMP proteolytic cleavage site has the amino acid sequence PLGLWA.

The proteolytic cleavage site may also comprise any aggrecanase specificcleavage site which is cleavable by an aggrecanase. An aggrecanasecleavage site may comprise a number of amino acid residues recognisableby an aggrecanase. Moreover, the amino acids of the aggrecanase site maybe linked by one or more peptide bonds which are cleavable,proteolytically, by aggrecanase.

Aggrecanases which may cleave the aggrecanase site include, but are notlimited to ADAMTS-4 (aggrecanase-1), ADAMTS-5 (aggrecanase-2) andADAMTS-11 (Tortorella, M. D., et al Osteoarthritis Cartilage, 2001.9(6): p. 539-552); Abbaszade, I., et al J Biol Chem, 1999. 274(33): p.23443-23450).

The sequences of the protein cleavage sites of ADAMTS-4 (aggrecanase-1)are shown in FIG. 3. Suitable ADAMTS-4 sites include:

(SEQ ID NO: 80) HNEFRQRETYMVF (SEQ ID NO: 97) DVQEFRGVTAVIR

The consensus ADAMTS-4 cleavage motif can be represented according toHills et al (J. Biol. Chem. 282 11101-11109 (2007)) as:

(SEQ ID NO: 129) E-[AFVLMY]-X-_((0,1))-[RK]-X_((2,3))-[ST]-[VYIFWMLA]

The aggrecanase proteolytic cleavage site of the present invention maybe cleaved by ADAMTS-4 (aggrecanase-1), ADAMTS-5 (aggrecanase-2) orADAMTS-11.

The amino acid sequence of the aggrecanase cleavage site may include asequence which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99%identity, using the default parameters of the BLAST computer programprovided by HGMP, thereto. Preferably, the nucleic acid sequenceencoding the aggrecanase cleavage site comprises the minimum number ofresidues required for recognition and cleavage by an aggrecanase.

The present invention may further provide a “linker” peptide. Preferablythe linker peptide is linked to the amino acid sequence of theproteolytic cleavage site. The linker peptide may be provided at the Cterminal or N terminal end of the amino acid sequence encoding theproteolytic cleavage site. Preferably, the linker peptide is continuouswith the amino acid sequence of the proteolytic cleavage site. Thelinker peptide may comprise the amino acid sequence GGGGS (SEQ IDNO:135) or a multimer thereof (for example a dimer, a trimer, or atetramer), a suitable linker may be (GGGGS)₃ (SEQ ID NO:136), or asequence of nucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95%or 99% identity, using the default parameters of the BLAST computerprogram provided by HGMP, thereto.

The term “linker peptide” is intended to define any sequence of aminoacid residues which preferably provide a hydrophilic region whencontained in an expressed protein. Such a hydrophilic region mayfacilitate cleavage by an enzyme at the proteolytic cleavage site.

The constructs of the invention may also comprise an additional linkersequence and/or proteolytic cleavage site between the dimerisationdomain and the LAP.

The term “latency” as used herein, may relate to a shielding effectwhich may hinder interaction between the fusion protein and othermolecules in the cell surface. Alternatively the term latency may beused to describe a reduction in the activity (up to and includingablation of activity) of a molecule/agent associated with the fusionprotein. The term latency may also relate to a stabilising effect of thefusion protein. The effect may be in full or partial, where a partialeffect is sufficient to achieve the latency of the active agent.

The term “associating with” in the context of the present invention isintended to include all means of association including, but not limitedto, chemical cross-linking or peptide bond linkage.

The pharmaceutically active agent may be a pharmaceutically activeprotein which can include, but is not limited to, an antibody, a growthfactor (e.g. TGFβ, epidermal growth factor (EGF), platelet derivedgrowth factor (PDGF), nerve growth factor (NGF), colony stimulatingfactor (CSF), hepatocyte growth factor, insulin-like growth factor,placenta growth factor); a differentiation factor; a cytokine e.g. aninterleukin, (e.g. IL1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29,IL-30, IL-31, IL-32 or IL-33 or an interferon (e.g. IFN-α, IFN-β andIFN-γ), tumour necrosis factor (TNF), IFN-γ inducing factor (IGIF), abone morphogenetic protein (BMP, e.g. BMP-1, BMP-2, BMP-3, BMP-4, BMP-4,BMP-5, BMP-6, BMP-7, BMP-8, BMP-9, BMP10, BMP-11, BMP-12, BMP-13); aninterleukin receptor antagonist (e.g. IL-1 ra, IL-1R11), a tumornecrosis factor inhibitor (TNF-R or anti-TNF); a chemokine (e.g. MIPs(Macrophage Inflammatory Proteins) e.g. MIPla and MIP113; MCPs (MonocyteChemotactic Proteins) e.g. MCP1, 2 or 3; RANTES (regulated uponactivation normal T-cell expressed and secreted)); a trophic factor; acytokine inhibitor; a cytokine receptor; a free-radical scavengingenzyme e.g. superoxide dismutase or catalase; a pro-drug convertingenzyme (e.g. angiotensin converting enzyme, deaminases, dehydrogenases,reductases, kinases, urate oxidase and phosphatases); a peptide mimetic;a protease inhibitor; a tissue inhibitor of metalloproteinases (TIMPse.g. TIMP1, TIMP2, TIMP3 or TIMP4) or a serpin (inhibitors of serineproteases). Preferably, the pharmaceutically active agent will bederived from the species to be treated e.g. human origin for thetreatment of humans.

Preferably, the pharmaceutically active agent may be a cytokine, e.g.IFNβ, IL-4, or IL-1ra, or a cytokine inhibitor, such as an antibody orantibody fragment, e.g. trastuzumab, and as defined herein below.

The interleukins and cytokines may be anti-inflammatory orpro-inflammatory. Anti-inflammatory cytokines and certain interleukins,such as IL-4 and/or IL-10, are suitable for the treatment of arthritis,whereas pro-inflammatory cytokines and other interleukins, such as IL-1and IL-2, are suitable for the treatment of cancer.

As used herein “peptide mimetics” includes, but is not limited to,agents having a desired peptide backbone conformation embedded into anon-peptide skeleton which holds the peptide in a particularconformation. Peptide mimetics, which do not have some of the drawbacksof peptides, are of interest in those cases where peptides are notsuitable in medicine.

Peptide mimetics may comprise a peptide backbone which is of the L- orD-conformation. Examples of peptides mimetics include melanocortin,adrenocorticotrophin hormone (ACTH) and other peptide mimetic agentswhich play a role in the central nervous system, endocrine system insignal transduction and in infection and immunity.

The pharmaceutically active agent may comprise a chemical compound suchas a chemotherapeutic agent or other synthetic drug. Alternatively, thepharmaceutically active agent may comprise an siRNA or a peptide nucleicacid (PNA) sequence e.g. a poly-lysine sequence which binds to nucleicacids and permeabilises lipid bilayers (Wyman et al., BiologicalChemistry, 379, 1045-1052 (1998)) or a KALA peptide which facilitatestransfer through lipid bilayers (Wyman et al., Biochemistry, 36,3008-3017 (1997)) or a protein transduction domain (PTD) that enablespolypeptides to enter cells via the plasma membrane (Pi et al MolecularTherapy 2, 339-347 (2000)).

The pharmaceutically active agent may be suitable for interacting withsoluble target molecules. Examples of soluble target molecules includecytokines, growth factors, signaling proteins and other ligands andreceptors.

The pharmaceutically active agent may be a cytokine inhibitor. The term“cytokine inhibitor” refers to a molecule that can block, reduce,inhibit or neutralise a function, an activity and/or the expression of acytokine. The cytokine inhibitor may be a protein (for example solublecytokine receptor protein); an antibody or antibody fragment; nucleicacid (for example siRNA or anti-sense nucleic acid) or organic orinorganic molecules.

Examples of suitable antibodies include but are not limited to anti-TNF(e.g. anti-TNF α, anti-TNF β), anti-interleukins (e.g. anti-IL-1,anti-IL-2, anti-IL-3, anti-IL-4, anti-IL-5, anti-II-6, anti-IL-7,anti-IL-8, anti-IL-9, anti-IL-10, anti-IL-11, anti-IL-12, anti-IL-13,anti-IL-14, anti-IL-15, anti-IL-16, anti-IL-17, anti-IL-18, anti-IL-19,anti-IL-20, anti-IL-21, anti-IL-22, anti-IL-23, anti-IL-24, anti-IL-25,anti-IL-26, anti-IL-27, anti-IL-28, anti-IL-29, anti-IL-30, anti-IL-31,anti-IL-32, anti-IL-33, anti-IL-34, anti-IL-35 and IL-36),anti-interferons (e.g. anti-INF-α, anti-INF-β, anti-INF-γ andanti-INF-ω) and fragments thereof.

Examples of such molecules also include trastuzumab (also known asHerclon™/Herceptin™), a monoclonal antibody to the HER2/neu receptor.

The pharmaceutically active agent may be an antibody or an antibodyfragment. An “antibody fragment” as referred to herein means any portionof a full length antibody. Examples of antibody fragments include, butare not limited to, Fab, Fab′, F(ab′)2, scFv, Fv, dsFv diabody and Fdfragments.

The term “single chain variable fragment” or “scFv” refers to an Fvfragment in which the heavy chain domain and the light chain domain arelinked. One or more scFv fragments may be linked to other antibodyfragments (such as the constant domain of a heavy chain or a lightchain) to form antibody constructs having one or more antigenrecognition sites.

The antibody or antibody fragment may be suitable for use in thetreatment of inflammatory conditions such as arthritis, gout,atherosclerosis, allograft rejection, Crohn's disease, inflammatorybowel disease, irritable bowel syndrome and colitis.

In an alternative embodiment, the invention further provides the fusionprotein of the present invention optionally in association with latentTGFβ binding protein (LTBP). Typically, the fusion protein is covalentlylinked to LTBP to form a complex. Preferably, the association ismediated by disulphide bond(s) between Cys No. 33 of LAP and the third 8Cys residue of LTBP. The LTBP associated with the fusion protein mayinclude, but is not limited to, LTBP 1, 2, 3 or 4 (Kanzaki et al., Cell,61, 1051-1061 (1990); Tsuji et al., Proc. Natl. Acad. Sci. USA, 87,8835-8839 (1990); Moren et al., J. Biol. Chem. 269, 32469-32478 (1994);Yin et al., J. Biol. Chem. 270, 10147-10160 (1995); Gibson et al., Mol.Cell. Biol. 15, 6932-6942 (1995); Saharinen et al., J. Biol. Chem. 273,18459-18469 (1998)), or fragments of LTBP such as that containing thethird 8 Cys repeat, or homologues having a sequence of amino acids ornucleotides which has at least 50%, 60%, 70%, 80%, 90%, 95% or 99%identity, using the default parameters of the BLAST computer programprovided by HGMP, to that of LTBP.

Cleavage of LTBP may release the fusion protein from the LTBP complex.Enzymes which may cleave LTBP in this manner include, but are notlimited to, thrombospondin (Schultz et al., The Journal of BiologicalChemistry, 269, 26783-26788 (1994); Crawford et al., Cell, 93, 1159-1170(1998)), transglutaminase (Nunes et al., J. Cell, Biol. 136, 1151-1163(1997); Kojima et al., The Journal of Cell Biology, 121, 439-448 (1993))MMP9 and MMP2 (Yu and Stamenkovic, Genes and Dev, 14, 163-176 (2000)).

The invention further provides nucleic acid encoding the fusion proteinof the first aspect of the invention as defined above. A second aspectof the invention provides a nucleic acid construct comprising a firstnucleic acid sequence encoding a pharmaceutically active agent, a secondnucleic acid sequence encoding a LAP and a third nucleic acid sequenceencoding a dimerisation domain polypeptide.

The term “nucleic acid construct” generally refers to any length ofnucleic acid which may be DNA, cDNA or RNA such as mRNA obtained bycloning or produced by chemical synthesis. The DNA may be single ordouble stranded. Single stranded DNA may be the coding sense strand, orit may be the non-coding or anti-sense strand. For therapeutic use, thenucleic acid construct is preferably in a form capable of beingexpressed in the subject to be treated.

The pharmaceutically active agent may be suitable for interacting withsoluble target molecules. Examples of soluble target molecules includecytokines, growth factors, signaling proteins and other ligands andreceptors.

In an embodiment of the invention, the first nucleic acid sequenceencodes a cytokine inhibitor. The term “cytokine inhibitor” refers to amolecule that can block, reduce, inhibit or neutralise a function, anactivity and/or the expression of a cytokine. The cytokine inhibitor maybe a protein (for example soluble cytokine receptor protein); anantibody or antibody fragment; nucleic acid (for example siRNA oranti-sense nucleic acid).

Where the first nucleic acid construct encodes an antibody or anantibody fragment, the antibody fragment may be, for example, an Fab,Fab′, F(ab′)2, scFv, Fv, dsFv diabody or Fd fragment. Examples of suchmolecules include trastuzumab (also known as Herclon™/Herceptin™), amonoclonal antibody to the HER2/neu receptor.

In some embodiments, the first nucleic acid sequence encodes the proteinIFNβ, IL-4 or IL-1ra. In one embodiment of the invention, the firstnucleic acid sequence encodes IFNβ, IL-4 or IL-1ra from a mouse or ahuman.

The nucleic acid construct of the second aspect of the invention may bein the form of a vector, for example, an expression vector, and mayinclude, among others, chromosomal, episomal and virus-derived vectors,for example, vectors derived from bacterial plasmids, frombacteriophage, from transposons, from yeast episomes, from insertionelements, from yeast chromosomal elements, from viruses such asbaculo-viruses, papova-viruses, such as SV40, vaccinia viruses,adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses,and vectors derived from combinations thereof, such as those derivedfrom plasmid and bacteriophage genetic elements, such as cosmids andphagemids. Generally, any vector suitable to maintain, propagate orexpress nucleic acid to express a polypeptide in a host, may be used forexpression in this regard. The vector may comprise a plurality of thenucleic acid constructs defined above, for example 2 or more.

The invention further provides a protein encoded by the nucleic acidconstruct of the second aspect of the invention optionally inassociation with latent TGFβ binding protein (LTBP) described herein.Typically, the protein encoded by the nucleic acid construct iscovalently linked to LTBP to form a complex. Preferably, the associationis mediated by disulphide bond(s) between Cys No. 33 of LAP and thethird 8 Cys residue of LTBP.

The nucleic acid construct of the second aspect of the inventionpreferably includes a promoter or other regulatory sequence whichcontrols expression of the nucleic acid. Promoters and other regulatorysequences which control expression of a nucleic acid have beenidentified and are known in the art. The person skilled in the art willnote that it may not be necessary to utilise the whole promoter or otherregulatory sequence. Only the minimum essential regulatory element maybe required and, in fact, such elements can be used to constructchimeric sequences or other promoters. The essential requirement is, ofcourse, to retain the tissue and/or temporal specificity. The promotermay be any suitable known promoter, for example, the humancytomegalovirus (CMV) promoter, the CMV immediate early promoter, theHSV thymidine kinase, the early and late SV40 promoters or the promotersof retroviral LTRs, such as those of the Rous Sarcoma virus (RSV) andmetallothionine promoters such as the mouse metallothionine-I promoter.The promoter may comprise the minimum comprised for promoter activity(such as a TATA element without enhancer elements) for example, theminimum sequence of the CMV promoter. Preferably, the promoter iscontiguous to the first and/or second nucleic acid sequence.

As stated herein, the nucleic acid construct of the second aspect of theinvention may be in the form of a vector. Vectors frequently include oneor more expression markers which enable selection of cells transfected(or transformed) with them, and preferably, to enable a selection ofcells containing vectors incorporating heterologous DNA. A suitablestart and stop signal will generally be present.

One embodiment of the invention relates to a cell comprising the nucleicacid construct of the second aspect of the invention. The cell may betermed a “host” cell, which is useful for the manipulation of thenucleic acid, including cloning. Alternatively, the cell may be a cellin which to obtain expression of the nucleic acid. Representativeexamples of appropriate host cells for expression of the nucleic acidconstruct of the invention include virus packaging cells which allowencapsulation of the nucleic acid into a viral vector; bacterial cells,such as Streptococci, Staphylococci, E. coli, Streptomyces and Bacillussubtilis; single cells, such as yeast cells, for example, Saccharomycescerevisiae, and Aspergillus cells; insect cells such as Drosophila S2and Spodoptera Sf9 cells, animal cells such as CHO, COS, C127, 3T3,PHK.293, and Bowes Melanoma cells and other suitable human cells; andplant cells e.g. Arabidopsis thaliana.

Introduction of an expression vector into the host cell can be affectedby calcium phosphate transfection, DEAE-dextran mediated transfection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Sambrook et al, Molecular Cloning, a Laboratory Manual, SecondEdition, Coldspring Harbor Laboratory Press, Coldspring Harbor, N.Y.(1989).

Mature proteins can be expressed in host cells, including mammaliancells such as CHO cells, yeast, bacteria, or other cells under thecontrol of appropriate promoters. Cell-free translation systems can beemployed to produce such proteins using RNAs derived from the nucleicacid construct of the second aspect of the present invention.Appropriate cloning and expression vectors for use with prokaryotic andeukaryotic hosts are described by Sambrook et al, Molecular Cloning, aLaboratory Manual, Second Edition, Coldspring Harbor Laboratory Press,Coldspring Harbor, N.Y. (1989).

Proteins can be recovered and purified from recombinant cell cultures bywell-known methods including ammonium sulphate or ethanol precipitation,acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography, highperformance liquid chromatography, lectin and/or heparin chromatography.For therapy, the nucleic acid construct e.g. in the form of arecombinant vector, may be purified by techniques known in the art, suchas by means of column chromatography as described in Sambrook et al,Molecular Cloning, a Laboratory Manual, Second Edition, ColdspringHarbor Laboratory Press, Coldspring Harbor, N.Y. (1989).

According to a third aspect of the invention, there is provided acomposition in accordance with the first aspect of the invention for usein the treatment of inflammatory conditions or cancer. This aspect ofthe invention therefore extends to and includes a method for thetreatment of inflammatory conditions or cancer comprising theadministration to a subject of a composition comprising a fusion proteincomprising a latency associated protein, a dimerisation domain and apharmaceutically active agent.

The present invention provides a composition as described above for usein the treatment of inflammatory conditions or cancer. Inflammatoryconditions include, without limitation, atherosclerosis, acute andchronic lung inflammation (e.g., chronic bronchitis, asthma, lunginfection including bacterial and viral infections such as SARS andinfluenza, cystic fibrosis, etc.), inflammation of virus-infectedtissues (e.g., viral lung infections, viral myocarditis, viralmeningitis, etc.), ulcerative colitis, endotoxic shock, arthritis (e.g.,rheumatoid arthritis, juvenile arthritis, osteoarthritis, psoriaticarthritis, reactive arthritis, viral or post-viral arthritis, ankylosingspondylarthritis, etc.), psoriasis, Crohn's disease, inflammatory boweldisease, insulin dependent diabetes mellitus, injury independent type IIdiabetes, ischemia induced inflammation, otitis media (middle earinfection), gout, multiple sclerosis, cachexia, and AtaxiaTelangiectasia. Arthritis defines a group of disease conditions (orarthropathies) where damage is caused to the joints of the body andincludes osteoarthritis (also known as degenerative joint disease) whichcan occur following trauma to the joint, following an infection of thejoint or as a result of aging. Other forms of arthritis includerheumatoid arthritis and psoriatic arthritis, which are autoimmunediseases, and septic arthritis is caused by infection in the joints.Cancer defines a group of diseases characterized by an abnormalproliferation of cells in the body, which can be defined as tumors, forexample glioma. Types of gliomas include ependymomas, astrocytomas,oligodendrogliomas and mixed gliomas. A Grade 4 astrocytoma is alsoknown as a glioblastoma.

In a fourth aspect, the invention provides a nucleic acid sequence inaccordance with the second aspect of the invention for use in thetreatment of inflammatory conditions or cancer. This aspect thereforeextends to and includes a method for the treatment of inflammatoryconditions or cancer comprising the administration to a subject anucleic acid construct of the second aspect of the invention. Where thenucleic acid construct is used in the therapeutic method of theinvention, the construct may be used as part of an expression construct,e.g. in the form of an expression vector such as a plasmid or virus. Insuch a method, the construct may be administered intravenously,intradermally, intramuscularly, orally or by other routes.

The nucleic acid construct of the second aspect of the invention, andproteins derived therefrom, may be employed alone or in conjunction withother compounds, such as therapeutic compounds, e.g. anti-inflammatorydrugs, cytotoxic agents, cytostatic agents or antibiotics. The nucleicacid constructs and proteins useful in the present invention arepreferably provided in an isolated form, and preferably are purified tohomogeneity.

As used herein, the term “treatment” includes any regime that canbenefit a human or a non-human animal. The treatment of “non-humananimals” extends to the treatment of domestic animals, including horsesand companion animals (e.g. cats and dogs) and farm/agricultural animalsincluding members of the ovine, caprine, porcine, bovine and equinefamilies. The treatment may be in respect of any existing condition ordisorder, or may be prophylactic (preventive treatment). The treatmentmay be of an inherited or an acquired disease. The treatment may be ofan acute or chronic condition. Preferably, the treatment is of acondition/disorder associated with inflammation. The first nucleic acidsequence of the nucleic acid construct of the third aspect of theinvention may encode a protein for use in the treatment of the disorder,including, but not limited to osteoarthritis, scleroderma, renaldisease, rheumatoid arthritis, inflammatory bowel disease, multiplesclerosis, atherosclerosis, cancer, or any inflammatory disease.

The nucleic acid construct of the second aspect of the invention may beused therapeutically in a method of the invention by way of genetherapy. Alternatively, protein encoded by the nucleic acid constructmay be directly administered as described herein.

Administration of the nucleic acid construct of the second aspect may bedirected to the target site by physical methods. Examples of theseinclude topical administration of the “naked” nucleic acid in the formof a vector in an appropriate vehicle, for example, in solution in apharmaceutically acceptable excipient, such as phosphate bufferedsaline, or administration of a vector by physical method such asparticle bombardment according to methods known in the art.

Other physical methods for administering the nucleic acid construct orproteins of the third aspect of the invention directly to the recipientinclude ultrasound, electrical stimulation, electroporation andmicroseeding. Further methods of administration include oraladministration or administration through inhalation.

Particularly preferred is the microseeding mode of delivery which is asystem for delivering genetic material into cells in situ in a patient.This method is described in U.S. Pat. No. 5,697,901.

The nucleic acid construct according to the second aspect of theinvention may also be administered by means of delivery vectors. Theseinclude viral delivery vectors, such as adenovirus, retrovirus orlentivirus delivery vectors known in the art. Other non-viral deliveryvectors include lipid delivery vectors, including liposome deliveryvectors known in the art.

Administration may also take place via transformed host cells. Suchcells include cells harvested from the subject, into which the nucleicacid construct is transferred by gene transfer methods known in the art.Followed by the growth of the transformed cells in culture and graftingto the subject.

As used herein the term “gene therapy” refers to the introduction ofgenes by recombinant genetic engineering of body cells (somatic genetherapy) for the benefit of the patient. Furthermore, gene therapy canbe divided into ex vivo and in vivo techniques. Ex vivo gene therapyrelates to the removal of body cells from a patient, treatment of theremoved cells with a vector i.e., a recombinant vector, and subsequentreturn of the treated cells to the patient. In vivo gene therapy relatesto the direct administration of the recombinant gene vector by, forexample, intravenous or intravascular means. Preferably the method ofgene therapy of the present invention is carried out ex vivo.

Preferably in gene therapy, the expression vector of the presentinvention is administered such that it is expressed in the subject to betreated. Thus for human gene therapy, the promoter is preferably a humanpromoter from a human gene, or from a gene which is typically expressedin humans, such as the promoter from human CMV.

For gene therapy, the present invention may provide a method formanipulating the somatic cells of human and non-human mammals.

The present invention also provides a gene therapy method which mayinvolve the manipulation of the germ line cells of a non-human mammal.

The present invention therefore provides a method for providing a humanwith a therapeutic protein comprising introducing mammalian cells into ahuman, the human cells having been treated in vitro to insert therein anucleic acid construct according to the second aspect of the invention.

Each of the individual steps of the ex vivo somatic gene therapy methodare also covered by the present invention. For example, the step ofmanipulating the cells removed from a patient with the nucleic acidconstruct of the third aspect of the invention in an appropriate vector.As used herein, the term “manipulated cells” covers cells transfectedwith a recombinant vector. Also contemplated is the use of thetransfected cells in the manufacture of a medicament for the treatmentof inflammatory conditions, such as arthritis or cancer, as definedherein above.

The present invention may also find application in veterinary medicinefor treatment/prophylaxis of domestic animals including horses andcompanion animals (e.g. cats and dogs) and farm animals which mayinclude mammals of the ovine, porcine, caprine, bovine and equinefamilies.

The present invention also relates to compositions comprising thenucleic acid construct or proteins of the first or second aspects of theinvention. Therefore, the fusion protein or nucleic acid constructs ofthe present invention may be employed in combination with thepharmaceutically acceptable carrier or carriers. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,liposomes, water, glycerol, ethanol and combinations thereof.

The pharmaceutical compositions of the invention may comprise two fusionproteins according to the first aspect of the invention, wherein thefusion proteins are associated at the dimerisation domain in each fusionprotein, or a nucleic acid sequence encoding two fusion proteinsaccording to the first aspect of the invention.

The pharmaceutical compositions may be administered in any effective,convenient manner effective for treating a patient's disease including,for instance, administration by oral, topical, intravenous,intramuscular, intranasal, or intradermal routes among others. Intherapy or as a prophylactic, the active agent may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

For administration to mammals, and particularly humans, it is expectedthat the daily dosage of the active agent will be from 0.01 mg/kg up to10 mg/kg body weight, typically around 1 mg/kg. The physician in anyevent will determine the actual dosage which will be most suitable foran individual which will be dependent on factors including the age,weight, sex and response of the individual. The above dosages areexemplary of the average case. There can, of course, be instances wherehigher or lower dosages are merited, and such are within the scope ofthis invention

References to uses of the fusions proteins, nucleic acid constructs,vectors, or host cells of the present invention in the treatment ofdiseases, such as inflammatory diseases or cancer, includes embodimentsrelating to the use of the fusion protein, nucleic acid construct,vector, or host cell in the manufacture of a medicament for thetreatment of said diseases.

A fifth aspect of the invention provides a fusion protein comprising aLAP, a pharmaceutically active agent and an amino acid sequencecomprising a dimerisation domain, wherein the LAP and thepharmaceutically active agent are connected by an amino acid sequencecomprising a proteolytic cleavage site for use in the treatment ofinflammatory conditions or cancer. The pharmaceutically active agent maybe as described above. In some embodiments of this aspect of theinvention, the pharmaceutically active agent may be an siRNA or PNAmolecule.

The invention further provides a nucleic acid construct encoding thefusion protein of the fifth aspect of the invention. The nucleic acidconstruct preferably comprises a nucleic acid sequence encoding a LAPadjacent a nucleic acid sequence encoding a proteolytic cleavage site.Preferably, the nucleic acid sequence encoding a LAP is suitablyoperably linked to a nucleic acid sequence encoding a proteolyticcleavage site.

The invention further provides the fusion protein of the fifth aspect ofthe invention optionally in association with latent TGFβ binding protein(LTBP) described herein.

The fusion protein of the fifth aspect of the invention may beassociated with the pharmaceutically active agent by means of a peptidebond linkage. Alternatively, the fusion protein may be associated withthe pharmaceutically active agent by means of a chemical linkage e.g. bycross-linking the fusion protein to a chemical compound such as achemotherapeutic agent, synthetic drug or PNA.

Preferably, the pharmaceutically active agent is linked to theC-terminal end of the amino acid sequence of the proteolytic cleavagesite in the fusion protein of the seventh aspect of the invention. Morepreferably, the pharmaceutically active agent is continuous with theC-terminal residue of the amino acid sequence of the proteolyticcleavage site.

The fusion protein, and associated pharmaceutically active agent of thefifth aspect of the invention may be employed alone or in conjunctionwith other compounds, such as therapeutic compounds, e.g.anti-inflammatory drugs, cytotoxic agents, cytostatic agents orantibiotics. Such administration may be simultaneous, separate orsequential. The components may be prepared in the form of a kit whichmay comprise instructions as appropriate.

Preferably, the fusion protein and associated pharmaceutically activeagent of the fifth aspect of the invention are directly administered toa patient as described herein.

The present invention also relates to compositions comprising the fusionprotein and associated pharmaceutically active agent of the fifth aspectof the invention. Therefore, the fusion protein and associatedpharmaceutically active agent may be employed in combination with thepharmaceutically acceptable carrier or carriers. Such carriers mayinclude, but are not limited to, saline, buffered saline, dextrose,liposomes, water, glycerol, polyethylene glycol, ethanol andcombinations thereof.

The pharmaceutical compositions may be administered in any effective,convenient manner effective for treating a disease of a patientincluding, for instance, administration by oral, topical, intravenous,intramuscular, intranasal, or intradermal routes among others. Intherapy or as a prophylactic, the active agent may be administered to anindividual as an injectable composition, for example as a sterileaqueous dispersion, preferably isotonic.

A sixth aspect of the invention provides a kit of parts comprising afusion protein of the first aspect of the invention, a nucleic acidconstruct of the second aspect of the invention, or a fusion protein andassociated pharmaceutically active agent according to the fifth aspectof the invention, and an administration vehicle including, but notlimited to, tablets for oral administration, inhalers for lungadministration and injectable solutions for intravenous administration.

A seventh aspect of the invention provides a process for preparing thefusion protein, of the first aspect of the invention comprisingproduction of the fusion protein recombinant by expression in a hostcell, purification of the expressed fusion protein and association ofthe pharmaceutically active agent to the purified fusion protein bymeans of peptide bond linkage, hydrogen or salt bond or chemical crosslinking. In some embodiments of this aspect of the invention where thepharmaceutically active agent is a peptide, the fusion protein could beprepared using hydrogen or salt bonds where the peptide is capable ormultimerisation, for example dimerisation or trimerisation.

An eighth aspect of the invention provides a process for preparing anucleic acid construct of the second aspect of the invention comprisingligating together nucleic acid sequences encoding a latency associatedpeptide, a proteolytic cleavage sequence, and a pharmaceutically activeagent, optionally including a linker sequence on either side of theproteolytic cleavage site.

One embodiment of the present invention provides a method of providinglatency to a pharmaceutically active agent which is a cytokine,preferably interferon or an interleukin or a cytokine inhibitor such asa scFV or soluble cytokine receptor, the method comprising constructinga fusion protein having a latency associated peptide, preferably fromTGFβ, associated with a proteolytic cleavage site, preferably anADAM-TS4 cleavage site, and the pharmaceutically active agent. Forexample, the pharmaceutically active agent may be followed by theproteolytic cleavage site and the LAP as follows: Ig-LAP-cleavagesite-active agent.

The present invention therefore enables the formation of a dimer whichsolves a problem of protein production in suspension cultures. Theinvention also enables (due to the closure of the “shell” of the LAPconstruct) the production of latent antibody fragments (e.g. inhibitorsof cytokine action) as therapeutic agents. Previously, the fusion of asingle peptide therapeutic agent with LAP left the “shell” partiallyopen and thus potentially exposed without the need for release byprotease action at the site of disease i.e. MMP/aggrecanase cleavage.For example, where the therapeutic peptide was a cytokine inhibitor,such as a scFv, it could interact if exposed with the cytokine beforerelease at the site of disease.

The present invention can use any type of immunoglobulin (Ig) from anyspecies (as their structure is extremely similar), but for certainclinical applications particular isotypes may be advantageous forexample for in mucosal tissues IgA may be used, for binding to Fcreceptors IgG2 may be used. Additionally, mutated forms of Fc thatprevent them from binding to Fc receptors or activating cell mediatedimmunity or complement activation could be used if it was desirable fora particular clinical application.

All preferred features of the second and subsequent aspects of theinvention are as for the first aspect mutatis mutandis.

The present invention will now be described by way of example only withreference to the accompanying figures wherein:

FIG. 1 shows amino acid sequences of the precursor domain of TGFβ1, 2and 3 (human, Hu) (SEQ ID NOs:1-3), TGFβ4 (chicken, Ck) (SEQ ID NO:4),TGFβ (frog, Fg) (SEQ ID NO:5). Arrows indicate the position of theproteolytic processing resulting in cleavage of the signal peptide ofTGFβ1 and of the mature TGFβs. N-linked glycosylation sites areunderlined, as is the integrin cellular recognition sequence (Robertsand Sporn, Peptide Growth Factors and their Receptors: Sporn, M B andRoberts, AB, Springer-Verlag, Chapter 8, 422 (1996)).

FIG. 2 shows the sequences of protein cleavage sites of matrixmetalloproteinases (MMPs) (Nagase and Fields, Biopolymers, 40, 399-416(1996)) (SEQ ID NOs:6-78).

FIG. 3 shows a multiple sequence alignment of the ADAMTS-4 epitopesequences with corresponding average percentage of phagemid cleavage andthe derived ADAMTS-4 cleavage motif. Predominant amino acids found at afrequency of greater than 40% in a particular position are illustratedwith a black background, in contrast to related amino acids which areshown with a grey background (reproduced from Hills et al J. Biol. Chem.282 11101-11109 (2007)) (SEQ ID NOs:79-129).

FIG. 4A shows the theoretical structure of LAP. FIG. 4B shows thetheoretical structure of Ig-LAP. FIG. 4C shows a schematicrepresentation of Ig-LAP.

FIG. 5 shows the DNA sequence (SEQ ID NO:130) and predicted amino acidsequence (SEQ ID NO:131) of mouse IgG1 (Fc)-LAP-MMP-IFN. Initiator ATGis at position 10 and stop codon at position 2025.

FIG. 6 shows the expression of Ig-LAP-IFN in CHO cells. Western blottingof LAP-IFN (slot 1) or Ig-LAP-IFN (slot 2). 20 μl supernatant fromsuspension CHO cells was run on 4-12% SDS-PAGE in non-denaturingconditions and blotted onto PDVF membrane. Then probed with goatanti-LAP antibodies. The bands were detected using HRP-conjugatedanti-goat antibody by chemiluminescence (ECL, Amersham) and exposed toautoradiography.

FIG. 7 shows the cleavage of Ig-LAP-IFN by MMP1. 20 μl of CHO cellsupernatant was incubated at 37° C. overnight without (slot 1) or withMMP1 (slot 2). The products were then run on a 4-12% SDS-PAGE gradientgel in non-denaturing conditions and blotted to a PDVF membrane.Anti-LAP antibodies were used to detect the uncleaved (slot 1) andcleaved product (slot 2). The molecular weight of the cleaved Ig-LAPcorresponds to the expected size of about 160 kDa.

FIG. 8 shows the DNA sequence of human Ig-LAP with anti-herNeu2(Herceptin™) antibody (SEQ ID NOs:132-134). The cleavage site in thisconstruct is an aggrecanase-specific site. The secretory signal peptideis derived from IL-2 (nucleotides 1-54), the human CH2 and CH3 domainsare derived from IgG1 (nucleotides 55-757) and is followed by a spacerwith unique restriction sites HindIII and Nco1 (nucleotides 758-771) andthe human LAP sequence (nucleotides 772-1515) is followed by theaggrecanase cleavage site which is flanked by GGGGS (SEQ ID NO:135)linkers (nucleotides 1516-1584) and finally the Herceptin™ scFv endingin a poly His tail (nucleotides 1585-2370).

FIG. 9 shows the detection of Herceptin™ by anti-His antibody labelledwith a fluorophore. Ig-LAP Herceptin™ was produced in CHO cells insuspension and the fusion protein was purified from cell supernatants byaffinity chromatography using a Protein A column. A fraction from thepurified material was applied to breast cancer cells expressing Her/neu2(SKBR3) or non-expressing (MDA-MD-231) before or after cleavage byaggrecanase. Herceptin™ scFv binds to the P185 Her Neu2 on breast cancercells only after aggrecanase release of Herceptin™ from the hulg-LAPfusion.

The invention is now described with reference to the followingnon-limiting examples:

Example 1: Cloning the IgG1 Fc Between the Signal Peptide of IL-2 andMouse LAP

An example of the preparation of a construct of the invention is asfollows. PCR of mouse IgG1 was used for cloning the IgG1 Fc into anEcoR1 site after the signal peptide of muLAP-MMP-IFN. The followingoligonucleotides were designed for linking in frame the coding regions.

Sense oligo: (SEQ ID NO: 143) 5′ ATG AAT TCC GGT TGT AAG CCTTGCATAAnti-sense oligo: (SEQ ID NO: 144) 5′GT GA ATT CCT CCA TGG AAG CTT TTT ACC AGG AGA GTG GGA GAG

The EcoR1 sites in the oligos are underlined and the Nco1 and HindIIIsites are in bold. These latter sites were introduced to allow fordirect cloning of additional MMP or aggrecanase cleavage sites. Becausethe IgG1 fragment could be inserted on the opposite orientation neededfor in-frame translation, the resulting clones were analysed byrestriction analysis and these containing the IgG1 fragment in the rightorientation sent for DNA sequencing. FIG. 5 depicts the DNA sequence ofa representative positive clone.

Ig-LAP-MMP-IFN is effectively secreted from CHO cells grown insuspension:

Supernatant from transiently transfected CHO cells was analysed afternon-reducing SDS-PAGE by western blotting using a goat anti-LAP antibody(R&D systems) (FIG. 6). The molecular weight of the Ig-LAP-IFN wasbigger than that of LAP-IFN (i.e. above 200 kDa as expected from aglycosylated dimerised protein).

Id-LAP-Fusion is Cleaved by Recombinant MMP1:

In order to establish whether the Ig-LAP-IFN fusion is still cleavableby MMP the inventors digested the protein in the CHO supernatant withrecombinant MMP-1 overnight at 37° C. The reaction was stopped with 25mM EDTA and then analysed by western blotting after non-denaturingSDS-PAGE (FIG. 7).

After Cleavage with MMP, Ig-LAP-IFN Releases IFN Biological Activity:

Mouse L929 cells were plated at 104 cells/well in 96 well plates. Thecells were incubated overnight with supernatants of Ig-LAP-IFNtransfected CHO cell cultures that was treated or untreated with MMP-1at double dilutions starting at 1:10. Then the medium was removed andthe cells were infected with encephalomyocarditis virus in a volume of50 μl for 16 hours as described (Adams et al. 2003). Cells were washedin PBS and 100 ml of Cell titer-Glo (Promega) cell lysis buffer. After20 minutes and room temperature, 50 μl were transferred to an opaqueplate and the luminescence (endogenous ATP levels) read in aLuminometer. 50% cell viability was assessed as half of luciferaseactivity compared to uninfected L929 cells (see Table 2 which shows MMPcleavage releases IFN activity from Ig-LAP-IFN).

TABLE 2 Luciferase IFN biological Treatment activity activity (U/ml)Control No infection 10973 N/A Control (+EMC) 64 Ig-LAP-IFN (no MMP1) +EMC 64 0 IgLAP-IFN (+MMP) + EMC 5068 30

Example 2: Human IQ-Human LAP with Anti-herNeu2 (Herceptin™) Antibody

Human Ig-human LAP with anti-herNeu2 (Herceptin™—Markiv et al. BMCBiotechnology 2011, 11:117) antibody construct was created with anaggrecanase-specific cleavage site in CHO cells in suspension (FIG. 8).The secretory signal peptide was derived from IL-2 (nucleotides 1-54),the human CH2 and CH3 domains were derived from IgG1 (nucleotides55-757). In the construct the human CH2 and CH3 domains are followed bya spacer with unique restriction sites HindIII and Nco1 (nucleotides758-771). The human LAP sequence (nucleotides 772-1515) is followed bythe aggrecanase cleavage site which is flanked by GGGGS linkers(nucleotides 1516-1584). The Herceptin™ scFv ends in a poly His tail(nucleotides 1585-2370). The fusion protein was purified from cellsupernatants by affinity chromatography using a Protein A column. Afraction from the purified material was applied to breast cancer cellsexpressing Her/neu2 (SKBR3) or non-expressing (MDA-MD-231) before orafter cleavage by aggrecanase. The bound Herceptin™ was detected byanti-His antibody labelled with a fluorophore (FIG. 9).

1-30. (canceled)
 31. A protein dimer comprising a pair of fusionproteins, each fusion protein comprising a latency associated peptide(LAP) which is the precursor domain of TGFβ-1, -2, -3, -4, or -5, apharmaceutically active agent and an amino acid sequence comprising adimerisation domain comprising an antibody fragment polypeptide, whereinthe LAP and the pharmaceutically active agent are connected by an aminoacid sequence comprising a proteolytic cleavage site, and thedimerisation domain is linked to the N-terminus of the LAP, and whereinthe fusion proteins are associated at the dimerisation domain in eachfusion protein and form a closed shell around the pharmaceuticallyactive agent, wherein the pharmaceutically active agent is an antibodyor antibody fragment.
 32. The protein dimer as claimed in claim 31,wherein the antibody fragment polypeptide is an Fc region polypeptide,an immunoglobulin hinge polypeptide, a CH3 domain polypeptide, a CH4domain polypeptide, a CH1 domain polypeptide, or a CL domainpolypeptide.
 33. The protein dimer as claimed in claim 31, wherein theantibody fragment polypeptide is an Fc region polypeptide.
 34. Theprotein dimer as claimed in claim 33, wherein the Fc region polypeptideis derived from an IgG or IgA antibody.
 35. The protein dimer as claimedin claim 31, wherein the dimerisation domain is linked to the latencyassociated peptide by a linker sequence.
 36. The protein dimer asclaimed in claim 31, wherein the proteolytic cleavage site is a matrixmetalloproteinase or an aggrecanase cleavage site.
 37. The protein dimeras claimed in claim 31, wherein the antibody fragment is an scFv, Fab,Fab′, F(ab′)₂, Fv, dsFv diabody, or Fd fragment.
 38. The protein dimeras claimed in claim 31, wherein the antibody or antibody fragmentthereof is an anti-TNF antibody, anti-interleukin antibody,anti-interferon antibody, anti-cytokine antibody, or fragment thereof.39. The protein dimer as claimed in claim 31, wherein the antibody orantibody fragment thereof is an antibody against the HER2/neu receptor.40. The protein dimer as claimed in claim 31, wherein the antibodyfragment is a trastuzumab scFv.
 41. A pharmaceutical compositioncomprising a protein dimer of claim
 31. 42. A nucleic acid constructencoding a fusion protein as defined in claim 31 comprising a nucleicacid sequence encoding a pharmaceutically active agent, a nucleic acidsequence encoding a LAP, and a nucleic acid sequence encoding adimerisation domain composed of an antibody fragment polypeptide,wherein the pharmaceutically active agent is an antibody or an antibodyfragment.
 43. The nucleic acid construct as claimed in claim 42, whereinthe dimerisation domain polypeptide is an Fc region polypeptide derivedfrom an IgG or IgA antibody.
 44. The nucleic acid construct as claimedin claim 42, wherein the nucleic acid construct further comprises anucleic acid sequence encoding a proteolytic cleavage site.
 45. Thenucleic acid construct as claimed in claim 42, which is in the form of avector.
 46. A fusion protein encoded by the nucleic acid construct ofclaim
 42. 47. A cell comprising a nucleic acid construct of claim 42.48. A pharmaceutical composition comprising a nucleic acid construct asclaimed in claim
 42. 49. A method for the treatment of an inflammatorycondition or cancer comprising the administration to a subject of acomposition comprising a protein dimer of claim
 31. 50. A method for thetreatment of an inflammatory condition or cancer comprising theadministration to a subject of a composition comprising a nucleic acidconstruct of claim
 42. 51. A kit comprising a protein dimer as claimedin claim 31 as an administration vehicle.
 52. A process for preparingthe protein dimer as claimed in claim 31, comprising producing thefusion proteins recombinantly by expression in a host cell, purifyingthe expressed fusion proteins, and associating the dimerisation domainof the fusion proteins at the N-terminus of the LAP to form a closedshell around the pharmaceutically active agent.
 53. A process forpreparing a nucleic acid construct of claim 42, comprising ligatingtogether nucleic acid sequences encoding a latency associated peptide, adimerisation domain, a proteolytic cleavage sequence, and apharmaceutically active agent, optionally including a linker sequence oneither side of the proteolytic cleavage site.